Downstream Targets Ribosomal Protein S6 Research Articles

IGF/mTORC1/S6 Signaling Is Potentiated and Prolonged by Acute Loading of Subtoxicological Manganese Ion.

The insulin-like growth factor (IGF)/insulin signaling (IIS) pathway is involved in cellular responses against intracellular divalent manganese ion (Mn2+) accumulation. As a pathway where multiple nodes utilize Mn2+ as a metallic co-factor, how the IIS signaling patterns are affected by Mn2+ overload is unresolved. In our prior studies, acute Mn2+ exposure potentiated IIS kinase activity upon physiological-level stimulation, indicated by elevated phosphorylation of protein kinase B (PKB, also known as AKT). AKT phosphorylation is associated with IIS activity; and provides direct signaling transduction input for the mammalian target of rapamycin complex 1 (mTORC1) and its downstream target ribosomal protein S6 (S6). Here, to better define the impact of Mn2+ exposure on IIS function, Mn2+-induced IIS activation was evaluated with serial concentrations and temporal endpoints. In the wild-type murine striatal neuronal line STHdh, the acute treatment of Mn2+ with IGF induced a Mn2+ concentration-sensitive phosphorylation of S6 at Ser235/236 to as low as 5 μM extracellular Mn2+. This effect required both the essential amino acids and insulin receptor (IR)/IGF receptor (IGFR) signaling input. Similar to simultaneous stimulation of Mn2+ and IGF, when a steady-state elevation of Mn2+ was established via a 24-h pre-exposure, phosphorylation of S6 also displayed higher sensitivity to sub-cytotoxic Mn2+ when compared to AKT phosphorylation at Ser473. This indicates a synergistic effect of sub-cytotoxic Mn2+ on IIS and mTORC1 signaling. Furthermore, elevated intracellular Mn2+, with both durations, led to a prolonged activation in AKT and S6 upon stimulation. Our data demonstrate that the downstream regulator S6 is a highly sensitive target of elevated Mn2+ and is well below the established acute cytotoxicity thresholds (<50 μM). These findings indicate that the IIS/mTORC1 pathways, in which Mn2+ normally serves as an essential co-factor, are dually responsible for the cellular changes in exposures to real-world Mn2+ concentrations.

Abstract B074: Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity

Abstract Objective: To determine the biological and signaling effects of dual-pathway inhibition with mTORC1/2 and MEK inhibitors in glioblastoma neurosphere cell lines. Background: The PI3K/mTOR signaling pathway is frequently dysregulated in glioblastoma (GBM), making it the focus of active scientific and drug development efforts in brain tumors. Unfortunately, small molecule targeted therapies against EGFR and its downstream effector pathways, while promising in the pre-clinical setting, have been disappointing in glioma clinical trials. The mTOR pathway target ribosomal protein S6 (RPS6) can also be activated by the ERK pathway target p90-RSK. We therefore tested the hypothesis that small molecule inhibition of both ERK and mTOR signaling pathways would result in enhanced anti-tumor activity. Methods: Seven glioblastoma neurosphere lines were tested for sensitivity to the ATP-competitive mTORC1/2 inhibitor sapanisertib (MLN0128) alone or in combination with the allosteric MEK1/2 inhibitor trametinib (GSK1120212) using cell growth assays and immunoblots as indicated. Results: The dual mTORC1/2 inhibitor sapanisertib inhibits mTOR signaling and cell growth in all GBM neurosphere lines, but only partially inhibits phosphorylation of the downstream target ribosomal protein S6 (phospho-RPS6) in these models. The MEK inhibitor, trametinib, inhibited ERK phosphorylation in all neurosphere lines, though phosphorylation of downstream targets and cell growth were inhibited to varying degrees. Inhibition of RPS6 activation was dose-dependent and correlated with cell line sensitivity when treated with trametinib but not with sapanisertib. In combination, sapanisertib plus trametinib showed more profound inhibition of phospho-RPS6, as well as enhanced growth inhibition and induction of apoptosis in sensitive neurosphere lines. Conclusions: The combination of sapanisertib and trametinib may have significant antitumor activity in glioblastoma neurospheres by enhanced inhibition of S6, which serves as the convergence point between these effector pathways. Citation Format: Karisa C Schreck, Amy Allen, Jiawan Wang, Christine A Pratilas. Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B074. doi:10.1158/1535-7163.TARG-19-B074

Cannabidiol Reduces Leukemic Cell Size - But Is It Important?

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.

Reciprocal regulation of mTOR complexes in pancreatic islets from humans with type 2 diabetes.

Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of nutritional status at the cellular and organismic level. While mTORC1 mediates beta cell growth and expansion, its hyperactivation has been observed in pancreatic islets from animal models of type 2 diabetes and leads to beta cell loss. We sought to determine whether such mTORC1 activation occurs in humans with type 2 diabetes or in metabolically stressed human islets and whether mTORC1 blockade can restore beta cell function of diabetic islets. Human islets isolated from non-diabetic controls and individuals with type 2 diabetes, as well as human islets and INS-1E cells exposed to increased glucose (22.2mmol/l), were examined for mTORC1/2 activity by western blotting analysis of phosphorylation of mTORC1 downstream targets ribosomal protein S6 kinase 1 (S6K1), S6 and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and mTORC2 downstream targets Akt and N-myc downstream regulated 1 (NDRG1). mTORC1/2 complexes' integrity was assessed by immunoprecipitation and subsequent western blot analysis. Cell-type specific expression of activated mTORC1 in human islets was examined by immunostaining of pS6 (Ser 235/236) in human islet sections. Beta cell function was measured by glucose-stimulated insulin secretion (GSIS). While mTORC2 signalling was diminished, mTORC1 activity was markedly increased in islets from patients with type 2 diabetes and in islets and beta cells exposed to increased glucose concentrations. Under high-glucose conditions in metabolically stressed human islets, we identified a reciprocal regulation of different mTOR complexes, with functional upregulation of mTORC1 and downregulation of mTORC2. pS6 immunostaining showed beta cell-specific upregulation of mTORC1 in islets isolated from patients with type 2 diabetes. Inhibition of mTORC1-S6K1 signalling improved GSIS and restored mTORC2 activity in islets from patients with type 2 diabetes as well as in islets isolated from diabetic db/db mice and mice fed a high-fat/high-sucrose diet. Our data show the aberrant mTORC1 activity in islets from patients with type 2 diabetes, in human islets cultured under diabetes-associated increased glucose conditions and in diabetic mouse islets. This suggests that elevated mTORC1 activation is a striking pathogenic hallmark of islets in type 2 diabetes, contributing to impaired beta cell function and survival in the presence of metabolic stress.

Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation.

Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.

Resistance to Selumetinib (AZD6244) in Colorectal Cancer Cell Lines is Mediated by p70S6K and RPS6 Activation

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Phase I study of UCN-01 and perifosine in patients with relapsed and refractory acute leukemias and high-risk myelodysplastic syndrome

The PI3K-Akt pathway is frequently activated in acute leukemias and represents an important therapeutic target. UCN-01 and perifosine are known to inhibit Akt activation. The primary objective of this phase I study was to determine the maximum tolerated dose (MTD) of UCN-01 given in combination with perifosine in patients with advanced acute leukemias and myelodysplastic syndrome. Secondary objectives included safety, pharmacokinetics, pharmacodynamics, and efficacy. Perifosine 150 mg every 6 h was given orally on day 1 followed by 100 mg once a day continuously in 28-day cycles. UCN-01 was given intravenously over 3 h on day 4 at three dose levels (DL1=40 mg/m(2); DL2=65 mg/m(2); DL3=90 mg/m(2)). Thirteen patients were treated (DL1, n=6; DL2, n=4; DL3, n=3) according to a traditional "3+3" design. Two patients at the DL3 experienced dose-limiting toxicity including grade 3-4 pericardial effusion, hypotension, hyperglycemia, hyperkalemia, constitutional symptoms and grade 5 pneumonitis. Other frequent toxicities were grade 1-2 nausea, diarrhea, vomiting, fatigue and hyperglycemia. The MTD was determined to be UCN-01 65 mg/m(2) with perifosine 100 mg a day. No appreciable direct Akt inhibition could be demonstrated in patients' mononuclear cells using Western blot, however, reduced phosphorylation of the downstream target ribosomal protein S6 in leukemic blasts was noted by intracellular flow cytometry. No objective responses were observed on this study. UCN-01 and perifosine can be safely administered, but this regimen lacked clinical efficacy. This approach may have failed because of insufficient Akt inhibition in vivo.

Phospho-Specific Flow Cytometry of Fixed Whole Blood During AML Clinical Trials to Monitor In Vivo FLT3 Inhibition in Leukemic Blasts,

Abstract 3508Although first-generation FLT3 inhibitors may have had limited anti-leukemic effects due to suboptimal target inhibition, newer drugs such as AC220 and KW-2449 have substantially greater in vitro potency and bioavailability. Ex vivo assays such as the plasma inhibition assay (PIA) are useful to estimate free drug bioavailability, but direct confirmation of biochemical FLT3 inhibition in leukemic blasts in vivo has proven more challenging to employ systematically for drug development. Here we report the development of a fixed whole blood intracellular flow cytometry platform to measure real-time signal inhibition during a clinical trial of the second-generation FLT3 inhibitor KW-2449. Methods:Anticoagulated blood samples were aliquoted into FACS tubes within four hours of collection; a subset was exposed to signaling inhibitors (KW-2449, rapamycin × 30 min.) or activators (phorbol ester/PMA or FLT3 ligand/FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed, red cells were lysed with the permeabilizing agent triton X-100, and specimens were stored at −20C in glycerol medium. Subjects’ samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single cocktail of antibodies. Blasts were identified by CD45 and side scatter (SSC) and confirmed by multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive gates for phospho-proteins were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results:Despite adequate controls, flow demonstrated limited changes in FLT3-ITD+ blasts' pSTAT5 signal following either FL stimulation or ex vivo KW-2449 treatment of these peripheral blood primary samples. This contrasted with the FLT3-ITD+ cell line Molm14, in which FLT3 inhibition reduced pSTAT5. However, the PI3K/AKT/mTOR downstream target ribosomal protein S6 (S6) was consistently observed to be constitutively phosphorylated in both Molm14 cells and peripheral blood FLT3-ITD+ AML blasts. pS6 in all FLT3-ITD+ samples markedly augmented with ex vivo FL, and decreased following ex vivo KW-2449 treatment. We therefore serially monitored S6 phosphorylation during therapy on a phase 1/2 trial of KW-2449.In this clinical trial, subjects were treated with KW-2449 every 6–8 hours, due to the drug’s relatively short half life. 10 subjects (9 FLT3-ITD+, 1 FLT3-WT) provided serial blood samples for analysis. All FLT3-ITD+ subjects had blasts identifiable by morphology and immunophenotype. Samples with as few as 500 blasts/uL were informative for pS6. In all cases, blasts showed dynamic changes in pS6 in response to ex vivo FL. As previously described using intracellular flow cytometry, pS6 in primary AML samples was heterogeneous, and, at basal state, frequently only demonstrable in a subset of blasts. We observed constitutive S6 phosphorylation in 8/9 subjects’ leukemic cells. The mean percentage of blasts with constitutive pS6 was 21% (median 7%, range 5–70%). To directly quantify FLT3 kinase inhibition in vivo, we serially monitored pS6 in blasts by flow prior to and following their initial oral KW-2449 dose. In 8/8 patients with baseline constitutive S6 phosphorylation, blood obtained two hours following the initial dose showed marked reduction in the percentage of pS6+ blasts to a mean of 3.8% (median 1.3% range 0.1 to 20%). This reflected an 83% mean reduction in the percentage of pS6+ blasts. PIA was performed in 8/9 of FLT3-ITD+ subjects and confirmed that potent FLT3-inhibitory concentrations were present 2 hours after a single dose of KW-2449 (mean reduction from baseline of 79% for pFLT3 and 88% for pSTAT5). Two subjects’ samples were followed serially by flow cytometry throughout the dosing interval. One showed sustained inhibition (consistent with concurrent PIA), while in the other, pS6 returned to baseline within 4–6 hours of the initial dose (concurrent PIA not done). Summary:We confirm that PI3K/AKT/mTOR is a major downstream pathway of FLT3 signaling in primary AML samples. We further demonstrate the feasibility of intracellular flow cytometry for S6 phosphorylation to monitor the biochemical efficacy of FLT3 inhibitors in patients. Studies are underway to correlate biochemical FLT3 inhibition by flow cytometry with clinical response/resistance to KW-2449 and other FLT3 inhibitors. Disclosures:Sato:Kyowa Hakko Kirin Co., LTD: Employment. Akinaga:Kyowa Hakko Kirin Co., LTD: Employment. Rao:Kyowa Hakko Kirin Co., LTD: Employment. Levis:Kyowa Hakko Kirin Co., LTD: Research Funding; Ambit Biosciences: Consultancy. Carroll:Kyowa Hakko Kirin Co., LTD: Research Funding.

Time course changes in signaling pathways and protein synthesis in C2C12myotubes following AMPK activation by AICAR

The role of the AMP-activated kinase (AMPK) as a metabolic sensor in skeletal muscle has been far better characterized for glucose and fat metabolism than for protein metabolism. Therefore, the studies presented here were designed to examine the effects of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of mRNA translation and protein synthesis in cultures of C(2)C(12) myotubes. The findings show that, following AICAR (2 mM) treatment, AMPK phosphorylation was increased within 15 min and remained elevated throughout a 60-min time course. In association with the increase in AMPK phosphorylation, global rates of protein synthesis declined to 90, 70, and 63% of the control values at the 15-, 30-, and 60-min time points, respectively. By 60 min, polysomes disaggregated into free ribosomal subunits, suggesting an inhibition of initiation of mRNA translation. However, phosphorylation of eukaryotic elongation factor 2 was increased at 15 and 30 min but then declined to control values by 60 min, suggesting a transient inhibition of translation elongation. The decline in protein synthesis and changes in mRNA translation were associated with a repression of the mammalian target of rapamycin (mTOR) signaling pathway, as indicated by increased association of Hamartin with Tuberin, increased association of regulatory associated protein of mTOR with mTOR, and dephosphorylation of the downstream targets ribosomal protein S6 kinase-1 and eukaryotic initiation factor 4E-binding protein-1. They were also associated with activation of the MAPK signaling pathway, as indicated by increased phosphorylation of MEK1/2 and ERK1/2 and the downstream target eIF4E. Overall, the data support the conclusion that AICAR-induced AMPK activation suppresses protein synthesis through concurrent repression of mTOR signaling and activation of MAPK signaling, the combination of which modulates transient changes in the initiation and elongation phases of mRNA translation.

A Potential Role for Extracellular Signal-regulated Kinases in Prostaglandin F2α-induced Protein Synthesis in Smooth Muscle Cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.